Salinomycin

ABSTRACT

Salinomycin, prepared by culturing Streptomyces albus has been found to be effective as an anticoccidium agent.

L nited States Patent 1191' Tanaka. et al.

[451 Dec. 31, 1974 SALINOMYCIN [75] Inventors: Yoshiaki Tanaka; HideoSaito, both of Tokyo; Yukio Miyazaki, Ageo; Hideo Sugawara, Saitama;Junsaku Nagatsu; Mitsuo Shibuya, both of Tokyo, all of Japan [73]Assignee: Kaken Chemical Co., Ltd., Tokyo,

Japan [22] Filed: Oct. 26, 1973 [21] Appl. No.: 410,030

3 Related U.S. Application Data [63] Continuation-impart of Ser. No.302,392, Oct. 31,

1972, abandoned;

[30] Foreign Application Priority Data Mar. 3, 1972 Japan 47-21553 OTHERPUBLlCATlONS Derwent, N0. 76960T-BD, Abstracting JA-47-25392, published10-20-72.

Primary ExaminerJer0me D. Goldberg Attorney, Agent, or Firm-Oblon,Fisher, Spivak, McClelland & Maier v [5 7 ABSTRACT Salinomycin, preparedby culturing Streptomyces albus has been found .to be effective as ananticoccidium agent.

3 Claims, 2 Drawing Figures SALINOMYCIN RELATIONSHIP TO RELATEDAPPLICATIONS This application is a continuation-in-part of application,Ser. No. 302,392, filed Oct. 31, 1972 now abandoned.

BACKGROUND OF THE INVENTION This invention relates to Salinomycin. Moreparticularly, it relates to an anticoccidium composition containingSalinomycin and an inactive carrier. Difficulty has arisen in thetreatment of coccidium, especially as it occurs in fowls such aschickens, because of the development of strains resistant to treatmentby conventional anticoccidium agents such as antithiamine and quinolineand sulfur containing agents. Therefore, a need exists for a newanticoccidium material for the treatment of resistant strains ofcoccidium.

SUMMARY OF THE INVENTION Accordingly, it is an object of this inventionto provide a preparation of Salinomycin from a Salinomycin producingmicroorganism.

It is another object of this invention to provide a new use ofSalinomycin as an anticoccidium agent and to provide an anticoccidiumcomposition.

This object and other objects of this invention, as hereinafter will bedescribed, can be attained by culturing Streptomyces albus 80,614 toform Salinomycinin a medium, isolating the Salinomycin by solventextraction of the mycelial cake and the filtrate of the liquid mediumand purifying it. The isolated Salinomycin is used as an anticoccidiumagent and is especially useful for preventive and therapeutic treatmentof fowls (chickens).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an ultraviolet spectrum ofSalinomycin in 0.01 N HCl,-methanol solution (undotted line) and 0.01 NNaOH-methanol solution (dotted line); and,

FIG. 2 is an infrared spectrum of Salinomycin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Salinomycin can be successfullyisolated from a fungus body and medium, which is cultured from aSalinomycin producing Streptomyces albus microorganism, Streptomycesalbus 80,614 (Fermentation Research Institute of the Agency ofIndustrial Science and Technology of Japan No. 419)ATCC 21,838.

Streptomyces albus 80,614 can be cultured by the same process employedfor Actinomycetes. For industrial operations, a stirred aerobic cultureprocess is preferred. The temperature of the culture is preferablymaintained from 25C. to 30C., and the culture medium can be any of theusual ones used for culturing Actinomycetes. The culture mediumcomprises a carbon source, a nitrogen source, inorganic salts, a smallamount of an organic component and an anti-foaming agent. The highestaccumulation of Salinomycin in the culture can be found in a period from72 120 hours after the initiation of culture.

Isolation ofSalinomycin can be accomplished by utilizing acharacteristic of Salinomycin. Salinomycin is soluble in various organicsolvents and can be isolated by a solvent extraction process. After anappropriate.

culturing period of Actinomycetes, the culture is til- Test tered andseparated into a mycelial cake and a filtrate, both of which containSalinomycin. The cake is treated with a solvent selected from the groupconsisting of butylacetate, acetone and chloroform, whereby the fungusbody is autolyzed and the Salinomycin in the fungus body is extracted.After combining the filtrate and the extract phase, the solutioncontaining Salinomycin is concentrated in vacuum and is purified in achromatographic column over alumina. The Salinomycin extract is elutedthrough the column with either ethyl acetate, hexane or a mixturethereof. The eluent is fractioned to yield fractions with a highantibiotic action. These fractions are concentrated in a vacuum andpurified through a column chromatograph containing a gel filteringmaterial, Sephadex LH-20. In this instance, Salinomycin is elutedthrough the column with acetone. The eluate is fractionated to collectactive fractions which are concentrated to yield powdery, purifiedSalinomycin. Streptomyces albus 80,614 has a long hypha which is notseparated into a bacillary or a coccoid. The aerial mycelium ofStreptomyces albus 80,614 is substantially straight, but sometimesdiverges to yield sporephores possessing 2 3 turns in a spiral form. Thesurface of the spore is smooth and it has no spine. The spore possessesa long ellipsoid or cylindrical shape of about 0.5 1.0 X 1.0 1.5g. insize.

The characteristics of Streptomyces albus 80,614 are as follows: I

Physiological reactions of Streptomyces albus 80,614

Response Milk coagulation Negative Milk peptonization Negative Melaninformation Negative Tyrosinase reaction Negative Nitrate reductionPositive, sometimes negative Starch hydrolysis Positive Liquefaction ofgelation Positive Decomposition of cellulose Negative ChromogenicityNegative Oxygen requirement Aerobic Optimum growing conditions pH 5.58.2

temperature .21 37C.-

Utilization pattern of carbon sources by Streptomyces albus 80,614(Pridham & Gottliebs basal medium) l-l-z glucose, fructose, galactose,mannitol, xylose, cellobiose, mebibiose, inulin +1 lactose, trehalose,starch i": maltose, mannose, sucrose, salicin, arabinose melezitose,inositol, dulcitol, sorbitol, raffinose, adonitol, rhamnose -ll-:strongly positive positive i: doubtfully positive negative Thephysico-chemical properties of Salinomycin (Na salt) are as follows:

1. Colorless, weak acidic powder:

Melting point 117 118C.

2. Optical rotation:

[01],, 25 (c 1, methanol) 3. Solubility: soluble in alcohols, esters,chloroform, ether, carbon tetrachloride and hexane; insoluble in water.

4. Stability: stable at pH 7 9; slightly unstable at a pH lower than 5.

5. Color reaction:

Negative for the Lemieux reaction, potassium per- Streptomyces albus80614 (ATCC 21,838) (PERM-P No. 419) was inoculated into 100 liters of aliquid medium containing 2% glucose, 1% starch, 2.5% soybean flour, 0.2%beer yeast, 0.2% meat extract and 0.2% sodium chloride, in a 200 literstainless steel tank at pH 7.0. The mixture was cultured at 27C. for 84hours under aerobic conditions. Air was passed into the culmanganatereaction, ninhydrin reaction, ferric 5 ture at the rate of 100liter/min. with stirring at the rate chloride reaction and Fehlingsreaction; a positive of 250 rpm. Upon termination of the culturingperiod, reddiSh-br0Wn Color for the iodine redetion; the resultingproduct was adjusted to pH 8 by the addi- 7 Ultraviolet spec r m: tionof NaOH, and further admixed with 2% diatoma- The uhdotted of 1represents the Spectrum ceous earth and filtered. An 83 liter quantityof the filofsahhomyeih measured in (1-01 N HC] methanol 10 trate wasadmixed with 50 liters of butyl acetate, and Solution, and the dottedhhe represents the'spee extracted two times. In addition, the fungusbody was t um measured in 0.01 N NaOH methanol S0luextracted with 30liters of 70% acetone. The acetone lion. solution was concentrated in avacuum and extracted 8. Infrared absorption spectrum: with 10 liters ofbutyl acetate after removing the ace- See tone. The butyl acetate wasadded to the filtrate and 9 Ahtimiemhial Spectra? the mixture was washedwith water and concentrated The minimum ihhihhory concentration g/ is ina vacuum to a 0.4 liter volume. The concentrated so- Stated n the Tablelution was passed through a column packed with 3 liters of activatedalumina to adsorb the components. A mixture of ethyl acetate andn-hexane (4 l) was Bacillus subulrs 2.0 3mg, 20 passed through thecolumn to elute the adsorbed mate- Bacljlhw rial, and the elutedfractions containing the active comgf fllf' f f iiflfg'fg 2 ponent wereconcentrated in a vacuum. The concen- Smphylvwvw epidermidis -0 tratedsolution was purified by a chromatographic colij' umn packed withSephadex LH-20 in which acetone icrococcus flavus 8.0 Micrococcus luteus4.0 was used as the eluting solution. The chromatographic g l g ATCC 607treatment was repeated twice to yield 8.5 g. of pure,

ycobacterzum phlei 4.0 Mycobaclerium avium 8.0 whlte P y salmomycm'Escherichia coli 100 Klebsiella pneumoniae W0 Use Of Salinomycin Proleu:vulgaris 100 Xanlhamonar oryzac 64 Salinomycin has been found to be aneffective treat- 2 ment for anticoccidium, especially as found in fowlsAllernaria kikuchiann 32 (chickens). Serious problems have evolved inthe prevention of coccidium in fowls (chickens), because of Duamycin,K-1 78, K-358, X-206, nigericin, Monensin 35 the development of strainsresistant to conventional an- A.B.C, and Dianemycin, and the like havecertain charti-coccidium agents, such as anti-thiamine, andquinoacteristics similar to those of Salinomycin. As exline and sulfurcontaining agents. Salinomycin has been pected, these antibiotics havedifferent melting points, found to be effective in the prevention andtherapeutic infrared spectra and elimentary analysis values.Salinotreatment of resistant and sensitive strains of coccidmycin hasultraviolet absorption maxima at 220 mp. ium. and 285 mu, when itsspectrum is measured in an The anti-coccidium composition of theinvention is acidic methanol solution. preferably prepared as aconcentrated composition by The Salinomycin of this invention has theformula, in admixing Salinomycin with a physiological non-toxic acidform: solid or liquid carrier to form a concentrate which can on cm on/cni /c1n 11i, !(|JlI ClI Gib-Cg; (I: C (in: OH OH cu, Lib-CH on c cn(JII' on: llOOC 211 111 on 0 oh o o (irr, o \CII;

\CH o ofi c bin JII3 C cfi crn $11. but

crn

which may be present in the form ofapharmaceutically be added to thefood and drink of fowls (chickens). acceptable salt or ester thereof.Suitable pharmaceuti- Suitable solid carriers include wheat flour,soybean cally acceptable salts are the salts of sodium, potasflour,rice, deoiled bran, talc, kaolin, chalk and diatosium, calcium.magnesium or ammonium. i able 415- maceous earth. Suitable liquidcarriers include physioters are the lower alkyl esters l-6) 0r henlylester logical salt solutions, distilled water and physiological C H O Na(molecular weight 772) (Natype) non-toxic organic solvents and the like.Other suitable CHHMOH (molecular Weight 750) (H type) additives includeemulsifiers, dispersing agents, suspending agents, wetting agents,concentration agents, Preparation of Salinomycin gelation agents, andactive ingredients including adhering accelerants such as soybean oil.

The anti-coccidium composition is preferably administered as a solid,and as previously mentioned, the carrier for the composition can be anorganic or an inorganic feed stuff. The active ingredient or compositionis mixed by stirring, shaking or crushing with growth promotingfeed-stuffs or feed-stuffs for layers or broilers (chickens) to form apowdery concentrate contain- 5 rate). ing a physiologically non-toxiccarrier. If the anti- The weights of the chickens were measured at thecoccidium composition is used as a liquid for oral time of inoculationand at the end of the test. Weight doses, Salimonycin is preferablydispersed in a physioincrease rates were calculated with the firstweight deslogically non-toxic carrier such as water. Other antiignatedas 100. coccidium ingredients may be added to the composib. O.P.G.(oocyst per gram of dung) tion in addition to a bird growth promotingingredient The O.P. G. was calculated from the Planktons and a growthpromoting ingredient for chicken eggs. counting board by observing thefeces 7 days after in- Preferably, the anti-coccidium compositioncontains 0Cl1latl0nmore than about 0.0001% by weight Salinomycin. IIdttOtI of feces The amount of Salinomycin required for the preven- Thecofldltlon of feces was Observed both mommg tive and therapeutictreatment of coccidium in fowls and f (chickens), can be in the rangefrom 0.001 0.05% by Dlsslecflon I weight. Coccidium is caused by Eimeriatanella and ef- The dlsease of h 'hteshhe was (Phserved and h fectivepreventive and therapeutic treatment of the dishuthber of oocysts the hh Jeluhum, Small ease can be achieved by using Salinomycin as followstestlne, and caecum were investigated. The numerals shown in Table IIIrepresent the number of infected Test 1 chickens, while the s mbols to+l+lcorres 0nd to 0 I p A group of the Highline species of male chickensrethe followmghohdmohs istant to 40 ppm of methylbenzoquate was infectedCaecum normal with the oocyst of Eimeria tenella in amounts of 7.2 XShape of the caecum h lh s l' 10 portions of oocyst per chicken. Theinfected chickl f of the caecum is S ty mud and yellow ens 14 daysnewborn) were separated into two groups. The feed stuff Show in Table Iwas admixed with Muscosol of the caecum lS partially dilated and is0.01% of the Salinomycin diluted with soybean meal. white The treatedmeal was fed to the first group of chickens, Shape of the caecumshubstamlahy nomlah while the second group of chickens was leftuntreated. Mglcusa of the caecum (mated Over of ace. TABLE I No blood isfound in the caecum.

Mucous is yellowish.

Fmnula of feed'stuff A small number of white dead spots and bleeding comflour 4181. spots are found in the mucosa of the caecum. TCfeed-stuff(wheatflour 85%) +l-+ Atrophy and deformation of the caecumare Soybean mm] 15%) 33 clearly found, and the length of the caecum isfish meal with soin i s g slightly longer than the rectum. Normalcontent of l i meal 3 40 the caecum is not found, and the caecum isfilled tricalcium phosphate 1 calcium Carin-mate O3 wlth blood clots ora gray cheese-like degenerated salt 0.45 material. lncrassation of thecaecum is remarkable 33E"; fi ig E 8'9 and it is breakable. Bleedingspots are sometimes inorganic component 0,1 found. Disease reaches thebase of the caecum but methionin 0.05 y not to h c I sf drug 0J4 +-l+l-Atrophy and deformation of the caecum are [00 clearly found. In general,the caecum is sausageshaped and its length is the same or shorter thanDoses of Salinomycin were continuously applied to the the rectum. Thedisease reaches into one-third to first group of chickens from the dayprior to infection one-fourth of the rectum. The other observations withthe oocyst to the end of the test. The oocyst was made are the same asthose of -Hl-.

TABLE II O.P.G. (X l0) Condition of feces I Group number weightmortality increase (death 7 8 9 rate rate) days days days eveevemornningning ing First group (Salinomycin) ll 106.4 8 0 0 0.32 normal feces(bloody feces were found for one but the chicken recovered) Second group(No Salinomycin) 10 91.2 I824 l4.88 0.32 soft feces were injected 15days after the birth of the chickens. All of the infected chickens weredissected 9 days after inoculation. The test results are shown in TablesII and III.

a. Weight increase rate and mortality rate (death found in all after 7days with bloody feces in about of the chickens.

TABLE III Dissecton of chickens (except those that died) Group Numdisease of caecum Number of ooeysts disease of the ber intestine other-H- -ilthen the caecum First group (Salinomycin) l 9 l 0 0 0 8 2 0 0none Second group (No Sa|inomycin) 3 0 0 O 3 0 0 0 1 2 none Salinomycinexhibited excellent therapeutic effects, as shown by the conditions ofthe feces, O.P.G. weight increase rate, death rate and dissectionobservations of the Salinomycin dosed group.

Test 2 A group of the White Leghorn species of newborn chickens wasinfected with the oocyst of Eimeria tenella in amounts of 5 X portionsof the oocyst per chicken. The chickens (21 days newborn) were separatedinto four groups. The feed-stuff shown in Table I was admixed withSalinomycin, and 0.001% Salinomycin was continuously administered to thefirst group starting from 5 days prior to the inoculation of the 00-TABLE IV The test results are shown in Tables IV and V. The tests werethe same as those summarized in Table I, and the numerals and symbolsare the same as those of Test 1.

Group Num- Amount of Weight mortality O.P.G. Condition ber activeincrease (death of ingredient rate rate) (7 days) feces First group 100.001 144.8 0 0 Normal feces (Salinomycin (meaty feces administered werefound in prior to one) inoculation) Second group (Salinomycin 10 0.01144.4 0 0 Normal feces administered from inoculation Third group(Reference) 10 0.01 137.7 20 2.1 X 10 Bloody feces were found in twoFourth group (Control) 10 135.2 11.8 X 10 Bloody and meaty feces werefound in all TABLE V Group Nbumdisease of caecum disease of theintestine other than the caecum -H- 4+? ++-ll- First group 10 9 l 0 O 0none Second group 10 8 2 0 O 0 none Third group 8 3 3 2 O 0 none Fourthgroup 4 0 0 1 3 0 none was administered to each group at variousconcentrations of Salinomycin. The results are shown in Table 10 incomparison to the group dosed with the known anticoccidium agentindicated.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many dissection observations of theSalinomycin dosed group- V], together with the referen e A f th bi d was5 changes and modifications can he made thereto with- 4 weeks old.

TABLE VI The effects of various concentrations of Salinomycin againstinfection with E.tenella Grou Drug concentration of drug No. of Durationof No. of Nov of O.P.G.*

No. in feed (ppm) infected medication birds dead oocysts (days) birds 1Salinomycin l 5 X10 8 l 4 3.3 X l0 2 do. l0 do. do. 10 7.8 X 3 do. do.do. 10 4 7.3 X 10 4 do do. do. 10 0 5.8 X 10 5 do. do. dov 1O 0 1.0 X 106 do. do. do. 10 0 8.7 X 10 7 clopidol 125 do. do 10 0 5.8 X 10 8infected control do. do. 10 7 7.9 X 10 9 uninfected I control do. do. l00 r 0 Daily oocyst counting was done for 8 days after the infection, andthe results are expressed as the number of oocysts per gram of feces.The figures shown present the maximum daily oocyst count.

TABLE VI out departing from the spiritor scope of the invention as setforth herein. cecal lesion Accordingly, what is claimed as new andintended to 1 be covered by Letters Patent is: I

30 1. An anti-coccidium composition which comprises l 3 3 4 an effectiveamount for treating coccidium of a Salinol g i Z mycin having theformula in acid form of: 4 5 3 2 and a pharmaceutical carrier. g g g 352. The anti-coccidium composition of claim 1, which 7 6 4 comprises aneffective amount for treating coccidium g 10 y i 2 7 of said Salinomycinand feed-stuff for fowl.

3. A process for treating coccidium in fowl which Salinomycin exhibitedexcellent therapeutic effects in combatting coccidium as shown by theconditions of the feces, O.P.G. weight increase rate, death rate andcomprises treating said infected fowl-with an effective amount of aSalinomycin sufficient to treat coccidium and a pharmaceutical-carrier,wherein said Salinomycin has the formula, in acid form of:

1. AN ANTI-COCCIDIUM COMPOSITION WHICH COMPRISES AN EFFECTIVE AMOUNT FORTREATING COCCIDIUM OF A SALINOMYCIN HAVING THE FORMULA IN ACID FORM OF:2. The anti-coccidium composition of claim 1, which comprises aneffective amount for treating coccidium of said Salinomycin andfeed-stuff for fowl.
 3. A process for treating coccidium in fowl whichcomprises treating said infected fowl with an effective amount of aSalinomycin sufficient to treat coccidium and a pharmaceutical carrier,wherein said Salinomycin has the formula, in acid form of: